Implantable mesh

ABSTRACT

An implantable mesh including demineralized bone fibers mechanically entangled into a biodegradable or permanent implantable mesh is provided. A method of preparing the implantable mesh is also provided. The method of preparing the implantable mesh includes mechanically entangling demineralized bone fibers with non-bone fibers to form the implantable mesh. The mechanical entanglement of the bone fibers into the implantable mesh is achieved by applying needle punching with barbed needles, spun lacing, entanglement with water jets or air jets or ultrasonic entanglement with ultrasonic waves. A method of implanting an implantable mesh at a target bone tissue site is also provided.

BACKGROUND

It is estimated that more than half a million bone grafting proceduresare performed in the United States annually with a cost over $2.5billion. These numbers are expected to double by 2020. Both natural boneand bone substitutes have been used as graft materials. Natural bone maybe autograft or allograft. Bone substitutes include natural or syntheticmaterials such as collagen, silicone, acrylics, calcium phosphate,calcium sulfate, or the like.

There are at least three ways in which a bone graft can help repair abone defect. The first is osteogenesis, the formation of new bone withinthe graft by the presence of bone-forming cells called osteoprogenitorcells. The second is osteoinduction, a process in which moleculescontained within the graft (e.g., bone morphogenic proteins and othergrowth factors) convert progenitor cells into bone-forming cells. Thethird is osteoconduction, a physical effect by which a matrix oftencontaining graft material acts as a scaffold on which bone and cells inthe recipient are able to form. The scaffolds promote the migration,proliferation and differentiation of bone cells for bone regeneration.

Bone fiber based-demineralized bone matrices for implantation exhibitimprovements in mechanical properties, including cohesiveness, fiberlength, fiber diameter or width, fiber aspect ratio, or a combination ofmultiple variables.

Demineralized bone matrices (DBMs) have been shown to exhibit theability to induce and/or conduct the formation of bone. It is thereforedesirable to implant and maintain a demineralized bone matrix at a sitewhere bone growth is desired. Some DBMs are available as putties, gels,pastes, or in some specific shapes, for example sheets. In some casesDBM sheets do not have sufficient integrity for some applications andcan be pulled apart when implanted into bone defects that are not easilyconfined. Oftentimes, when DBM fibers are made they lack cohesivenessand tend to fall apart or become loose in the package or duringprocessing. In order to reduce this tendency, a carrier (for example,glycerol) is commonly added to keep the DBM fibers together. Theinclusion of a carrier can lead to additional manufacturing expenses andfurther complicate regulatory approval processes.

Therefore, there is a need for DBM compositions and methods that allowosteogenesis, osteoinduction and/or osteoconduction and at the sametime, have sufficient integrity to wrap around a bone defect, helpcontain other allograft, autograft and/or synthetic graft material andcan accept sutures therein without fear of tearing apart. DBMcompositions and methods that can be reinforced by a resorbable ornon-resorbable mesh that do not need a carrier would be beneficial.Furthermore, reinforced DBM compositions and methods that easily allowhydration of the demineralized bone matrix would also be beneficial.

SUMMARY

An implantable mesh including demineralized bone fibers mechanicallyentangled into the implantable mesh is provided. In some embodiments,the implantable mesh comprises a demineralized bone fiber mechanicallyentangled with a biodegradable mesh fiber; in other embodiments, theimplantable mesh comprises a plurality of biodegradable mesh fibersmechanically entangled with each other that are then mechanicallyentangled with one or more demineralized bone fibers; in yet otherembodiments, the implantable mesh comprises biodegradable fibersmechanically entangled with the demineralized bone fibers to form aplurality of layers in the implantable mesh.

In various embodiments, the implantable mesh further includes anosteinductive and/or osteopromotive additive including a bone marrowaspirant, blood, a blood product, a bone morphogenetic protein, a growthfactor disposed on the biodegradable mesh fiber. In certain embodiments,the implantable mesh further includes a therapeutic agent or mixturesthereof. In other aspects, the implantable mesh includes collagen fibersmechanically entangled into the mesh. In certain embodiments, theimplantable mesh can include non-bone fibers, for example, chips,shards, particles and/or shavings, which are of a sufficientconfiguration to become mechanically entangled with the biodegradable orpermanent mesh.

In certain embodiments, the bone fibers become mechanically entangledinto the implantable mesh by needle punching with barbed needles,entanglement with water or air jets, ultrasonic entanglement withultrasonic waves. In other embodiments, the implantable mesh containingdemineralized bone fibers mechanically entangled into it is furthersubjected to moisture, heat and/or pressure provided by pressurerollers. In some embodiments, the implantable mesh containingmechanically entangled demineralized bone fibers is lyophilized.

In various embodiments, the implantable mesh containing demineralizedbone fibers mechanically entangled therein includes autograft orallograft bone. In some embodiments, the implantable mesh containingdemineralized bone fibers mechanically entangled therein contains wovenor nonwoven bone fibers. The demineralized bone fibers can have anaspect ratio of from about 50:1 to about 1000:1, from about 50:1 toabout 950:1, from about 50:1 to about 750:1, from about 50:1 to about500:1, from about 50:1 to about 250:1, from about 50:1 to about 100:1,from about 10:1 to about 50:1, or from about 5:1 to about 10:1. In someembodiments, the demineralized bone fibers have a diameter from about100 μm to about 2 mm. In other embodiments, the demineralized bonefibers have a length from about 0.5 cm to about 10 cm.

In certain embodiments, a method of preparing an implantable mesh isprovided. The method comprises mechanically entangling demineralizedbone fibers with non-bone fibers to form the implantable mesh. In otherembodiments, the mechanical entangling comprises applying to thedemineralized bone fibers and the non-bone fibers needle punching withbarbed needles, spun lacing, entanglement with water jets or air jets orultrasonic entanglement with ultrasonic waves. In various aspects, theimplantable mesh does not contain a carrier or adhesive.

In some embodiments, a method of treating a target bone tissue site isprovided. The method of treatment includes contacting the bone tissuesite with the implantable mesh, the implantable mesh comprisingdemineralized bone fibers mechanically entangled into the implantablemesh. In other embodiments, the method of treatment further includescontacting the implantable mesh with a liquid and molding themechanically entangled demineralized bone material into a shape which isconfigured to fit at, near or in the target bone tissue site. The liquiduseful for contacting the implantable mesh containing demineralized bonefiber mechanically entangled therein, in various aspects, includesphysiologically acceptable water, physiological saline, sodium chloride,dextrose, Lactated Ringer's solution, phosphate buffered saline, blood,bone marrow aspirate, bone marrow fractions or a combination thereof inan amount sufficient to render the implantable osteogenic materialmoldable. In some embodiments, the implantable mesh is configured to bewrapped around the target bone tissue site. In other embodiments, theimplantable mesh can be secured with sutures to the target bone tissuesite without tearing upon removal.

Additional features and advantages of various embodiments will be setforth in part in the description that follows, and in part will beapparent from the description, or may be learned by practice of variousembodiments. The objectives and other advantages of various embodimentswill be realized and attained by means of the elements and combinationsparticularly pointed out in the description and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments will be apparent with regard to the following description,appended claims and accompanying drawings where:

FIG. 1 depicts two barbed needles used in needle punching technology;

FIG. 2 depicts a schematic of a needle punching process;

FIG. 3 depicts a schematic of a spunlaced or hydroentanglement process;and

FIG. 4A depicts a side view of DBM fibers. FIG. 4B illustrates a sideview of reinforcement mesh. FIG. 4C is a side view of an implant meshresulting from mechanically entangling the DBM fibers of FIG. 4A intothe Mesh of FIG. 4B.

FIG. 5A depicts an example of DBM sheets/shavings containing naturalcollagen fibers. FIG. 5B depicts a mesh that can be used to reinforcethe DBM sheets/shavings of FIG. 5A. FIG. 5C illustrates an implant meshresulting from mechanically entangling the DBM sheets/shavings of FIG.5A into the mesh of FIG. 5B.

It is to be understood that the figures are not drawn to scale. Further,the relation between objects in a figure may not be to scale, and may infact have a reverse relationship as to size. The figures are intended tobring understanding and clarity to the structure of each object shown,and thus, some features may be exaggerated in order to illustrate aspecific feature of a structure.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thedisclosure, reference will now be made to certain embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alterations and furthermodifications in the illustrated bone material, and such furtherapplications of the principles of the disclosure as described hereinbeing contemplated as would normally occur to one skilled in the art towhich the disclosure relates.

Additionally, unless defined otherwise or apparent from context, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs.

Unless explicitly stated or apparent from context, the following termsare phrases have the definitions provided below:

Definitions

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities of ingredients,percentages or proportions of materials, reaction conditions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about.”Similarly, when values are expressed as approximations, by use of theantecedent “about,” it will be understood that the particular valueforms another embodiment that is +/−10% of the recited value.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Also, as used inthe specification and including the appended claims, the singular forms“a,” “an,” and “the” include the plural, and reference to a particularnumerical value includes at least that particular value, unless thecontext clearly dictates otherwise. Ranges may be expressed herein asfrom “about” or “approximately” one particular value and/or to “about”or “approximately” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of this application are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a range of “1 to 10” includes any and allsubranges between (and including) the minimum value of 1 and the maximumvalue of 10, that is, any and all subranges having a minimum value ofequal to or greater than 1 and a maximum value of equal to or less than10, e.g., 5.5 to 10.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “an allograft” includes one, two, three or more allografts.

The terms “bioactive” composition or “pharmaceutical” composition asused herein may be used interchangeably. Both terms refer tocompositions that can be administered to a subject. Bioactive orpharmaceutical compositions are sometimes referred to herein as“pharmaceutical compositions” or “bioactive compositions” of the currentdisclosure.

The term “biodegradable” includes that all or parts of the carrierand/or implant will degrade over time by the action of enzymes, byhydrolytic action and/or by other similar mechanisms in the human body.In various embodiments, “biodegradable” includes that the carrier and/orimplant can break down or degrade within the body to non-toxiccomponents after or while a therapeutic agent has been or is beingreleased. By “bioerodible” it is meant that the carrier and/or implantwill erode or degrade over time due, at least in part, to contact withsubstances found in the surrounding tissue, fluids or by cellularaction.

The term “mammal” refers to organisms from the taxonomy class“mammalian” including, but not limited to, humans; other primates suchas chimpanzees, apes, orangutans and monkeys; rats, mice, cats, dogs,cows, horses, etc.

A “therapeutically effective amount” or “effective amount” is such thatwhen administered, the drug (e.g., growth factor) results in alterationof the biological activity, such as, for example, promotion of bone,cartilage and/or other tissue (e.g., vascular tissue) growth, inhibitionof inflammation, reduction or alleviation of pain, improvement in thecondition through inhibition of an immunologic response, etc. The dosageadministered to a patient can be as single or multiple doses dependingupon a variety of factors, including the drug's administeredpharmacokinetic properties, the route of administration, patientconditions and characteristics (sex, age, body weight, health, size,etc.), and extent of symptoms, concurrent treatments, frequency oftreatment and the effect desired. In some embodiments the implant isdesigned for immediate release. In other embodiments the implant isdesigned for sustained release. In other embodiments, the implantcomprises one or more immediate release surfaces and one or moresustained release surfaces.

The terms “treating” and “treatment” when used in connection with adisease or condition refer to executing a protocol that may include abone repair procedure, where the bone implant and/or one or more drugsare administered to a patient (human, other normal or otherwise or othermammal), in an effort to alleviate signs or symptoms of the disease orcondition or immunological response. Alleviation can occur prior tosigns or symptoms of the disease or condition appearing, as well asafter their appearance. Thus, treating or treatment includes preventingor prevention of disease or undesirable condition. In addition,treating, treatment, preventing or prevention do not require completealleviation of signs or symptoms, does not require a cure, andspecifically includes protocols that have only a marginal effect on thepatient.

The term “bone,” as used herein, refers to bone that is cortical,cancellous or cortico-cancellous of autogenous, allogenic, xenogenic, ortransgenic origin.

The term “allograft” refers to a graft of tissue obtained from a donorof the same species as, but with a different genetic make-up from, therecipient, as a tissue transplant between two humans.

The term “autologous” refers to being derived or transferred from thesame individual's body, such as for example an autologous bone marrowtransplant.

The term “osteoconductive,” as used herein, refers to the ability of anon-osteoinductive substance to serve as a suitable template orsubstance along which bone may grow.

The term “osteoinductive,” as used herein, refers to the quality ofbeing able to recruit cells from the host that have the potential tostimulate new bone formation. Any material that can induce the formationof ectopic bone in the soft tissue of an animal is consideredosteoinductive.

The term “osteoinduction” refers to the ability to stimulate theproliferation and differentiation of pluripotent mesenchymal stem cells(MSCs). In endochondral bone formation, stem cells differentiate intochondroblasts and chondrocytes, laying down a cartilaginous ECM, whichsubsequently calcifies and is remodeled into lamellar bone. Inintramembranous bone formation, the stem cells differentiate directlyinto osteoblasts, which form bone through direct mechanisms.Osteoinduction can be stimulated by osteogenic growth factors, althoughsome ECM proteins can also drive progenitor cells toward the osteogenicphenotype.

The term “osteoconduction” refers to the ability to stimulate theattachment, migration, and distribution of vascular and osteogenic cellswithin the graft material. The physical characteristics that affect thegraft's osteoconductive activity include porosity, pore size, andthree-dimensional architecture. In addition, direct biochemicalinteractions between matrix proteins and cell surface receptors play amajor role in the host's response to the graft material.

In other instances, osteoinduction is considered to occur throughcellular recruitment and induction of the recruited cells to anosteogenic phenotype. Osteoinductivity score refers to a score rangingfrom 0 to 4 as determined according to the method of Edwards et al.(1998) or an equivalent calibrated test. In the method of Edwards etal., a score of “0” represents no new bone formation; “1” represents1%-25% of implant involved in new bone formation; “2” represents 26-50%of implant involved in new bone formation; “3” represents 51%-75% ofimplant involved in new bone formation; and “4” represents >75% ofimplant involved in new bone formation. In most instances, the score isassessed 28 days after implantation. However, the osteoinductivity scoremay be obtained at earlier time points such as 7, 14, or 21 daysfollowing implantation. In these instances it may be desirable toinclude a normal DBM control such as DBM powder without a carrier, andif possible, a positive control such as BMP. Occasionally,osteoinductivity may also be scored at later time points such as 40, 60,or even 100 days following implantation. Percentage of osteoinductivityrefers to an osteoinductivity score at a given time point expressed as apercentage of activity, of a specified reference score. Osteoinductivitymay be assessed in an athymic rat or in a human. Generally, as discussedherein, an osteoinductive score is assessed based on osteoinductivity inan athymic rat.

The term “osteogenic” refers to the ability of a graft material toproduce bone independently. To have direct osteogenic activity, thegraft must contain cellular components that directly induce boneformation. For example, an allograft seeded with activated MSCs wouldhave the potential to induce bone formation directly, withoutrecruitment and activation of host MSC populations. Because manyosteoconductive allografts also have the ability to bind and deliverbioactive molecules, their osteoinductive potential will be greatlyenhanced.

The term “osteoimplant,” as used herein, refers to any bone-derivedimplant prepared in accordance with the embodiments of this disclosureand, therefore, is intended to include expressions such as bone membraneor bone graft. Osteoimplant is used herein in its broadest sense and isnot intended to be limited to any particular shapes, sizes,configurations, compositions, or applications. Osteoimplant refers toany device or material for implantation that aids or augments boneformation or healing. Osteoimplants are often applied at a bone defectsite or bone cavity, for example, one resulting from injury, defectbrought about during the course of surgery, infection, malignancy,inflammation, or developmental malformation. Osteoimplants can be usedin a variety of orthopedic, neurosurgical, dental, and oral andmaxillofacial surgical procedures such as the repair of simple andcompound fractures and non-unions, external, and internal fixations,joint reconstructions such as arthrodesis, general arthroplasty, deficitfilling, disectomy, laminectomy, anterior cervical and thoracicoperations, or spinal fusions.

The term “patient” refers to a biological system to which a treatmentcan be administered. A biological system can include, for example, anindividual cell, a set of cells (e.g., a cell culture), an organ, or atissue. Additionally, the term “patient” can refer to animals,including, without limitation, humans.

The term “demineralized,” as used herein, refers to any materialgenerated by removing mineral material from tissue, e.g., bone tissue.In certain embodiments, the demineralized compositions described hereininclude preparations containing less than 5%, 4%, 3%, 2% or 1% calciumby weight. Partially demineralized bone (e.g., preparations with greaterthan 5% calcium by weight but containing less than 100% of the originalstarting amount of calcium) is also considered within the scope of thedisclosure. In some embodiments, partially demineralized bone containspreparations with greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% of the original starting amount of calcium. In some embodiments,demineralized bone has less than 95% of its original mineral content. Insome embodiments, demineralized bone has less than 95%, 90%, 85%, 80%,75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or5% of its original mineral content. Demineralized is intended toencompass such expressions as “substantially demineralized,” “partiallydemineralized,” and “fully demineralized.” In some embodiments, part orthe entire surface of the bone can be demineralized. For example, partor the entire surface of the allograft can be demineralized to a depthof from about 100 to about 5000 microns, or about 150 microns to about1000 microns. In some embodiments, part or all of the surface of theallograft can be demineralized to a depth of from about 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500,1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100,2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700,2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300,3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900,3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500,4550, 4600, 4650, 4700, 4750, 4800, 4850, 4900, 4950 to about 5000microns. If desired, the outer surface of the intervertebral implant canbe masked with an acid resistant coating or otherwise treated toselectively demineralize unmasked portions of the outer surface of theintervertebral implant so that the surface demineralization is atdiscrete positions on the implant.

The term “demineralized bone matrix,” (DBM) as used herein, refers toany material generated by removing mineral material from bone tissue. Insome embodiments, the DBM compositions as used herein includepreparations containing less than 5%, 4%, 3%, 2% or 1% calcium byweight. In other embodiments, the DBM compositions comprise partiallydemineralized bone (e.g., preparations with greater than 5% calcium byweight but containing less than 100% of the original starting amount ofcalcium) are also considered within the scope of the currentapplication. DBM preparations have been used for many years inorthopedic medicine to promote the formation of bone. For example, DBMhas found use in the repair of fractures, in the fusion of vertebrae, injoint replacement surgery, and in treating bone destruction due tounderlying disease such as a bone tumor. DBM has been shown to promotebone formation in vivo by osteoconductive and osteoinductive processes.The osteoinductive effect of implanted DBM compositions results from thepresence of active growth factors present on the isolated collagen-basedmatrix. These factors include members of the TGF-R, IGF, and BMP proteinfamilies. Particular examples of osteoinductive factors include TGF-β,IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenicfactors. Other osteoinductive factors such as osteocalcin andosteopontin are also likely to be present in DBM preparations as well.There are also likely to be other unnamed or undiscovered osteoinductivefactors present in DBM.

The term “superficially demineralized,” as used herein, refers tobone-derived elements possessing at least about 90, 91, 92, 93, 94, 95,96, 97, 98 or 99 weight percent of their original inorganic mineralcontent. The expression “partially demineralized” as used herein refersto bone-derived elements possessing from about 8 to about 90 weightpercent of their original inorganic mineral content. In someembodiments, partially demineralized refers to bone-derived elementspossessing from about 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88 to about 90 weight percent oftheir original inorganic mineral content. The expression “fullydemineralized” as used herein refers to bone containing less than 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1% of its original mineral context.

The terms “pulverized bone”, “powdered bone” or “bone powder” as usedherein, refers to bone particles of a wide range of average particlesize ranging from relatively fine powders to coarse grains and evenlarger chips.

The allograft can comprise bone fibers. Fibers include bone elementswhose average length to average thickness ratio or aspect ratio of thefiber is from about 50:1 to about 1000:1. In overall appearance thefibrous bone elements can be described as elongated bone fibers,threads, narrow strips, or thin sheets. Often, where thin sheets areproduced, their edges tend to curl up toward each other. The fibrousbone elements can be substantially linear in appearance or they can becoiled to resemble springs. In some embodiments, the elongated bonefibers are of irregular shapes including, for example, linear,serpentine or curved shapes. The elongated bone fibers aredemineralized, in some embodiments, however, some of the originalmineral content may be retained when desirable for a particularembodiment. The fibers when wet relax because they are porous, as theydry, they become more entangled and can be mechanically entangled toform a coherent mass as the fibers interconnect. In some embodiments,even when the fibers are wet, they are still cohesive.

“Non-fibrous,” as used herein, refers to elements that have an averagewidth substantially smaller than the average thickness of the fibrousbone element or aspect ratio of less than from about 50:1 to about1000:1. For example, allograft bone fibers will have a fiber shape,while the non-fibrous material will not have a fiber shape but will havea shape such as, for example, triangular prism, sphere, cube, cylinder,square, triangle, particle, powder, and other regular or irregularshapes.

“Pressed bone fibers,” as used herein, refer to bone fibers formed byapplying pressure to bone stock. The bone utilized as the starting, orstock, material may range in size from relatively small pieces of boneto bone of such dimensions as to be recognizable as to its anatomicalorigin. The bone may be substantially fully demineralized, surfacedemineralized, partially demineralized, or nondemineralized. In general,the pieces or sections of whole bone stock can range from about 1 toabout 400 mm, from about 5 to about 100 mm, in median length, from about0.5 to about 20 mm, or from about 2 to about 10 mm, in median thicknessand from about 1 to about 20 mm, or from about 2 to about 10 mm, inmedian width. Forming bone fibers by pressing results in intact bonefibers of longer length than other methods of producing the elongatebone fibers retaining more of the native collagen structure. The bonefibers may be made via a cartridge mill.

“High porosity,” as used herein refers to having a pore structure thatis conducive to cell ingrowth, and the ability to promote cell adhesion,proliferation and differentiation.

“Resorbable,” as used herein, refers to a material that exhibitschemical dissolution when placed in a mammalian body.

“Bioactive agent” or “bioactive compound,” as used herein, refers to acompound or entity that alters, inhibits, activates, or otherwiseaffects biological or chemical events. For example, bioactive agents mayinclude, but are not limited to, osteogenic or chondrogenic proteins orpeptides, anti-AIDS substances, anti-cancer substances, antibiotics,immunosuppressants, anti-viral substances, enzyme inhibitors, hormones,neurotoxins, opioids, hypnotics, anti-histamines, lubricants,tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants including channelblockers, miotics and anti-cholinergics, anti-glaucoma compounds,anti-parasite and/or anti-protozoal compounds, modulators ofcell-extracellular matrix interactions including cell growth inhibitorsand antiadhesion molecules, vasodilating agents, inhibitors of DNA, RNAor protein synthesis, anti-hypertensives, analgesics, anti-pyretics,steroidal and non-steroidal anti-inflammatory agents, anti-angiogenicfactors, angiogenic factors, anti-secretory factors, anticoagulantsand/or antithrombotic agents, local anesthetics, ophthalmics,prostaglandins, anti-depressants, anti-psychotic substances,anti-emetics, and imaging agents. In certain embodiments, the bioactiveagent is a drug. In some embodiments, the bioactive agent is a growthfactor, cytokine, extracellular matrix molecule or a fragment orderivative thereof, for example, a cell attachment sequence such as RGDpeptide.

“Coherent mass,” as used herein, refers to a plurality of bone fibers,in some embodiments, bound to one another by mechanical entanglement ofthe fibers. The cohesive mass may be in a variety of shapes and sizes,and is implantable into a surgical location. The cohesive mass comprisesat least two bone fibers, in some aspects, curled or partially curledbone fibers that entangle with one another to maintain a connectionwithout the use of a binding agent or carrier. In some embodiments, thefibers when wet relax because they are porous, as they dry, they becomemore entangled and form a coherent mass as the fibers interconnect.

The term “implantable” as utilized herein refers to a biocompatibledevice (e.g., implant) retaining potential for successful placementwithin a mammal. The expression “implantable device” and expressions ofthe like import as utilized herein refers to an object implantablethrough surgery, injection, or other suitable means whose primaryfunction is achieved either through its physical presence or mechanicalproperties. An example of the implantable device is the osteoimplant.

Localized delivery includes delivery where one or more implants aredeposited within a tissue, for example, a bone cavity, or in closeproximity (within about 0.1 cm, or in some aspects, within about 10 cm,for example) thereto.

Particle refers to pieces of a substance of all shapes, sizes, thicknessand configuration such as fibers, threads, narrow strips, thin sheets,clips, shards, etc., that possess regular, irregular or randomgeometries. It should be understood that some variation in dimensionwill occur in the production of the particles and particlesdemonstrating such variability in dimensions are within the scope of thepresent application. For example, the mineral particles (e.g., ceramic)can be from about 0.5 mm to about 3.5 mm. In some embodiments, themineral particles can be from about 0.2 mm to about 1.6 mm.

In some embodiments, the coherent mass of mechanically entangleddemineralized bone fibers forms a matrix. The “matrix” of the presentapplication is utilized as a scaffold for bone and/or cartilage repair,regeneration, and/or augmentation. Typically, the matrix provides a 3-Dmatrix of interconnecting pores, which acts as a scaffold for cellmigration. The morphology of the matrix guides cell migration and cellsare able to migrate into or over the matrix, respectively. The cellsthen are able to proliferate and synthesize new tissue and form boneand/or cartilage. In some embodiments, the matrix is resorbable.

In some embodiments, the matrix can be malleable, cohesive, flowableand/or can be shaped into any shape. The term “malleable” includes thatthe matrix is capable of being converted from a first shape to a secondshape by the application of pressure.

The term “cohesive” as used herein means that the mechanically entangleddemineralized bone fibers tend to remain a singular, connected mass uponmovement, including the exhibition of the ability to elongatesubstantially without breaking upon stretching.

The term “moldable” includes that the matrix can be shaped by hand ormachine or injected in the target tissue site (e.g., bone defect,fracture, or void) in to a wide variety of configurations. In someembodiments, the matrix can be formed into sheets, blocks, rings,struts, plates, disks, cones, pins, screws, tubes, teeth, bones, portionof bone, wedges, cylinders, threaded cylinders, or the like, as well asmore complex geometric configurations.

Reference will now be made in detail to certain embodiments of thedisclosure. The disclosure is intended to cover all alternatives,modifications, and equivalents that may be included within thedisclosure as defined by the appended claims.

The headings below are not meant to limit the disclosure in any way;embodiments under any one heading may be used in conjunction withembodiments under any other heading.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to various embodimentsdescribed herein without departing from the spirit or scope of theteachings herein. Thus, it is intended that various embodiments coverother modifications and variations of various embodiments within thescope of the present teachings.

Mesh Material

Suitable mesh materials include natural materials, synthetic polymericresorbable materials, synthetic polymeric non-resorbable materials, andother materials. Natural mesh materials include silk, extracellularmatrix (such as DBM, collagen, ligament, tendon tissue, or other),silk-elastin, elastin, collagen, and cellulose. Synthetic polymericresorbable materials include poly(lactic acid) (PLA), poly(glycolicacid) (PGA), poly(lactic acid-glycolic acid) (PLGA), polydioxanone, PVA,polyurethanes, polycarbonates, polyhydroxyalkanoates(polyhydroxybutyrates and polyhydroxyvalerates and copolymers),polysaccharides, polyhydroxyalkanoates polyglycolide-co-caprolactone,polyethylene oxide, polypropylene oxide, polyglycolide-co-trimethylenecarbonate, poly(lactic-co-glycolic acid), and others. See Chen and Wu,“The Application of Tissue Engineering Materials,” Biomaterials, 2005,26(33): p. 6565-78, herein incorporated by reference in its entirety.Other suitable materials include carbon fiber, metal fiber,polyertheretherketones, non-resorbable polyurethanes, polyethers of alltypes, polyethylene terephthalate, polyethylene, polypropylene, Teflon,and various other meshes. In other embodiments, the mesh may comprisenon-woven material such as spun cocoon or shape memory materials havinga coil shape or shape memory alloys.

In some embodiments, woven material and braided material can be includedin the mesh that can be used in this application. For example, U.S. Pat.No. 8,740,987, herein incorporated by reference in its entiretydiscloses tissue-derived mesh for orthopedic regeneration. In otherembodiments, the mesh can include non-woven materials, shape memorymaterial, porous materials and non-porous materials. In yet otherembodiments, outer particles may be used to contain inner particles,particles may be attached to threads of material, and/or porosity may beadded to mesh fibers.

In some embodiments, mesh fibers may be treated to impart porosity tothe fibers. This may be done, for example, to PLA, PLGA, PGA, and otherfibers. The implantable mesh is porous having pores from about 100 toabout 200 μm.

One suitable method for treating the mesh fibers comprises supercriticalcarbon dioxide treatment to partially solubilize the particles. Thistreatment may further be carried out for viral inactivation. Anothersuitable method for treating the mesh fibers comprises explosivedecompression. Explosive decompression generates porosity and leads tocontrolled permeability. The mesh material further may be loaded withcells, growth factors, or bioactive agents.

In further embodiments, fibers of a mesh material may be treated byhaving particles adhered thereto. The particles may be, for example,bone particles. Thus, in one embodiment, the mesh may comprise aplurality of threads formed into a fabric. The threads may haveparticles adhered thereto. For example, the threads may have particlesstrung on the thread. In an alternative embodiment, the mesh may beformed of a material and the material may be coated with particles.

In yet other embodiments, the mesh may comprise a non-porous material,which may be permeable. A non-porous material may be used for later (ordelayed) delivery of a substance provided therein. Such substance maycomprise, for example, cells, growth factors, or bone morphogeneticproteins. Accordingly, in one embodiment, a delivery system for delayeddelivery of cells, growth factors, or bone morphogenetic proteins isprovided comprising a non-porous mesh.

In particular, in various embodiments, the mesh may comprise abioerodible, a bioabsorbable, and/or a biodegradable biopolymer that mayprovide immediate release, or sustained release of an additive, in someaspects, a bioactive agent. Examples of suitable sustained releasebiopolymers include but are not limited to poly(alpha-hydroxy acids),poly(lactide-co-glycolide) (PLGA), polylactide (PLA), polyglycolide(PG), polyethylene glycol (PEG) conjugates of poly(alpha-hydroxy acids),poly(orthoester)s (POE), polyaspirins, polyphosphagenes, collagen,starch, pre-gelatinized starch, hyaluronic acid, chitosans, gelatin,alginates, albumin, fibrin, vitamin E compounds, such as alphatocopheryl acetate, d-alpha tocopheryl succinate, D,L-lactide, orL-lactide, -caprolactone, dextrans, vinylpyrrolidone, polyvinyl alcohol(PVA), PVA-g-PLGA, PEGT-PBT copolymer (polyactive), PEO-PPO-PAAcopolymers, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407,PEG-PLGA-PEG triblock copolymers, SAIB (sucrose acetate isobutyrate) orcombinations thereof. Useful biodegradable synthetic polymers include,for example, polylactic acid, polyglycolide, polylactic polyglycolicacid copolymers (PLGA), polycaprolactone (PCL), poly(dioxanone),poly(trimethylene carbonate) copolymers, polyglyconate, poly(propylenefumarate), poly(ethylene terephthalate), poly(butylene terephthalate),polyethylene glycol, polycaprolactone copolymers, polyhydroxybutyrate,polyhydroxyvalerate, tyrosine-derived polycarbonates and any random or(multi-)block copolymers, such as bipolymer, terpolymer, quaterpolymer,that can be polymerized from the monomers related to previously-listedhomo- and copolymers. Useful materials to be incorporated into the meshmay include, for example, natural polymers such as proteins andpolypeptides, glycosaminoglycans, proteoglycans, elastin, hyaluronicacid, dermatan sulfate, gelatin, or mixtures or composites thereof.Synthetic polymers may also be incorporated into the bone graftcomposites.

As persons of ordinary skill are aware, mPEG and/or PEG may be used as aplasticizer for PLGA, but other polymers/excipients may be used toachieve the same effect. mPEG imparts malleability to the resultingformulations.

In some embodiments, these biopolymers may also be coated on the mesh toprovide the desired release profile. In some embodiments, the coatingthickness may be thin, for example, from about 5, 10, 15, 20, 25, 30,35, 40, 45 or 50 microns to thicker coatings 60, 65, 70, 75, 80, 85, 90,95, 100 microns to delay release of the substance from the medicaldevice. In some embodiments, the range of the coating on the mesh rangesfrom about 5 microns to about 250 microns or 5 microns to about 200microns to delay release from the mesh. In various embodiments, themedical device comprises poly(lactide-co-glycolide) (PLGA), polylactide(PLA), polyglycolide (PGA), D-lactide, D,L-lactide, L-lactide,D,L-lactide-co-ε-caprolactone,D,L-lactide-co-glycolide-co-ε-caprolactone, L-lactide-co-ε-caprolactoneor a combination thereof.

Generally, the mesh may be formed as a sheet. In some embodiments, thematerial may be a textile type material. Thus, for example, the mesh maybe formed using a textile approach such as weaving, rug making orknitting. Such formation may be by a mechanical or industrial method. Inanother embodiment, a substantially solid sheet may be formed and may betreated to assume a configuration penetrable by cells, fluids, andproteins. For example, the sheet may be perforated, may be expanded tocreate openings, or otherwise make it penetrable by cells, fluids andproteins.

In one embodiment, elongated bone-derived particles or fragments ofsmall intestinal submucosa may be combined longitudinally into threesmall bundles, each having, for example, from about 1 to about 3 tissueparticles. The three bundles may then be braided. Various methods ofbraiding and types of braids any of which may be useful in producing themesh useful in this application. The ends of the braided tissue-derivedparticles may then be glued together using a fixation agent to preventtheir unraveling, or they may be held together with a biocompatiblepolymer or metal band.

In an alternative embodiment, bone-derived particles are combined with asolvent to form a material that can be used to generate threads forweaving into a mesh. Exemplary solvents include water, lower alkanols,ketones, and ethers and mixtures of any of these or other materials. Thematerial may then be extruded at an appropriate temperature and pressureto create a thread. Threads may also be produced by spinning, drawing,rolling, solvent-extruding, cutting or laser cutting from a sheet or barstock. The material may alternatively be cast or molded into a solidsheet or bar stock and then cut into thin threads. These may be usedimmediately or woven into a mesh. Alternatively, or in addition, theymay be spliced, wrapped, plied, cabled, braided, woven, or somecombination of these. The material may be shaped by thermal or chemicalbonding, or both. In one embodiment, a portion of the solvent is removedfrom the material before extrusion.

Alternatively or in addition, the material may be cast as a slurry,extruded, or molded. A variety of materials processing methods will bewell known to those skilled in the art. For example, the material may besolvent cast using a press such as a Carver press to spread the materialinto a film. Solvent evaporation will yield a porous film.Alternatively, the material may be compression molded into a film. Themesh size or porosity of the film will depend on the thickness of thefilm and the viscosity of the precursor and can be easily manipulated byone skilled in the art. Where elongated particles are used in anextruded aggregate, they will tend to be aligned roughly parallel to oneanother.

In an alternative embodiment, a thread of a biocompatible natural orsynthetic material, for example, polylactide or collagen, may be coatedwith tissue-derived or other elements, for example, by dubbing. Forexample, a polymer fiber may be coated with an adhesive, for example,lecithin, and bone particles or other osteoconductive or osteoinductivefibrils allowed to adhere to the thread. The thread may then be twistedon itself or with a second or a plurality of similarly treated threads.Alternatively, or in addition, the threads may be braided. The adhesivemay be a lipid that is waxy at room temperature, for example, a di- ortri-glyceride that is solid at room temperature. Alternatively, or inaddition, the adhesive may be a phosphocholine or phosphatidylcholine.In some embodiments, the adhesive is a material that binds both thethread and the material that is used to coat the thread (e.g., boneparticles) but that does not degrade either. Non-aqueous adhesives mayimprove the stability of the final aggregate as compared to aqueousadhesives.

Suitable fibers may be formed utilizing well known techniques, includingbraiding, plying, knitting, weaving, felting, that are applied toprocessing natural fibers, for example, cotton, silk, and syntheticfibers made from synthetic bioabsorbable polymers, such aspoly(glycolide) and poly(lactic acid), nylon, cellulose acetate. In someembodiments, collagen thread is wound onto cylindrical stainless steelspools. The spools are then mounted onto the braiding carousel, and thecollagen thread is then assembled in accordance with the instructionsprovided with the braiding machine. In one particular run, a braid wasprepared of four collagen threads, which consisted of two threads ofnon-crosslinked collagen and two threads of crosslinked collagen. Oneskilled in the art will recognize that these techniques may be appliedto the other fibrous materials described herein.

Fibers and more evenly dimensioned particles may also be plied intoyarns using the same methods and same machinery known to those skilledin the art in plying threads made out of other material, such as cottonor polyester. Four collagen threads were twisted together. Three of theresultant 4-ply strands were then twisted together in the oppositedirection, and then 5 of the resultant 12 ply strands were twisted inthe opposite direction.

Elongated materials including multistranded materials, for example,braids, plied yarns or cables, may be knitted into tubular or flatfabrics by using techniques known to those skilled in the art ofproducing fabrics manufactured from other types of threads. Variousbiologically active substances can be incorporated in, or associatedwith, the braided, knitted, or woven materials. Particles and fibers andmaterials of these (including multistranded materials) may alternativelyor additionally be assembled into a material by non-woven methods suchas laying, needle-punching, and hooking (as for a rug). For example, athread may be attached to another thread or a pressed film.

Regardless of the assembly method, the material shape, mesh size, cablethickness, and other structural characteristics, such as architecture,may be customized for the desired application. For example, where a twodimensional aggregate is used to retain a thixotropic material within agap, a tight weave is used, in some aspects, to prevent leakage. Tooptimize cell or fluid migration through the mesh, the pore size may beoptimized for the viscosity and surface tension of the fluid or the sizeof the cells. For example, pore sizes on the order of approximately fromabout 100 μm to about 200 μm may be used if cells are to migrate throughthe implantable mesh. Mesh size may be controlled by physically weavingstrands of the material by controlling the ratio of solvent to solids ina precursor material.

Cells may be seeded onto the mesh, or contained within it. In oneembodiment, cells may be encapsulated in a matrix such as alginate orcollagen gel and the capsules placed on the material. Seeded materialsgenerally do not need to be incubated for long periods of time insolutions that could partially dissolve the binding agent. Instead, thecapsules may be placed on the mesh shortly before implantation. Inanother embodiment, cells are simply mixed with a gel which is thencombined with the mesh. Alternatively, the mesh may be cultured withcells before implantation. In one embodiment, thicker materials are usedfor culturing to increase mechanical integrity during implantation. Anyclass of cells, including connective tissue cells, organ cells, musclecells, nerve cells, and stem cells, may be seeded onto the implant. Inan exemplary embodiment, connective tissue cells such as osteoblasts,osteoclasts, fibroblasts, tenocytes, chondrocytes, and ligament cellsand partially differentiated stem cells such as mesenchymal stem cellsand bone marrow stromal cells are employed.

Bone Material

DBM compositions and methods that allow osteogenesis, osteoinductionand/or osteoconduction are provided. DBM compositions and methods areprovided that allow osteogenesis, osteoinduction and/or osteoconduction.The DBM compositions and methods provided, in some embodiments, are madefrom bone material that does not contain or require a carrier in orderto stay in place during a surgical procedure and are also irrigationresistant. DBM compositions, devices and methods that easily allowhydration of the demineralized bone matrix are also provided.

Bone can be milled into fibers, shavings, sheets, prior to or afterdemineralization. Demineralized bone also naturally contains collagenfibers of various lengths depending on the milling/cutting process.

In some embodiments, demineralized bone fibers can be milled and formedinto mats with random fiber orientation. Subsequently, in other aspects,the demineralized bone fiber mats can be bonded together by applyingmoisture, heat and pressure created by pressure rollers so that thedemineralized bone fibers form a nonwoven sheet of matted fibers.

In other embodiments, the demineralized bone fibers can be furthermechanically entangled by additional mechanical means, such as needlepunching, spun-lace entanglement or by applying ultrasonic waves. Insome embodiments, felting needles can engage demineralized bone fibersand mechanically entangle them into a mesh sheet to permanentlytransport bundles of fibers into the mesh sheet to create a coherentfibrous structure of demineralized bone fibers mechanically entangledinto an implantable mesh.

In various embodiments, the felting needles can be forked or barbed andare used to hook the fibers to perform a fiber entanglement function.There are many variations in needle design, barb placement, barb angleand barb shape. FIG. 1 illustrates two embodiments of barbed needledesign. Each needle 10 and 12 include a crank 14, a shank 16, in oneaspect with an intermediate blade 18, a tip blade 24 and a point 26.Each needle can have the same or a different barb placement. Forexample, needle 10 has barbs 20 at an angle that is different andopposite in direction to the angle of the barbs 22 of needle 12.

FIG. 2 is a simplified schematic of a process of forming an implantablemesh comprising, consisting essentially of, or consisting ofdemineralized bone fibers mechanically entangled into a biodegradable ornon-biodegradable mesh by utilizing a conventional needle punching orfelting apparatus 100. Generally, a needle punching or felting apparatusincludes a needle board 102 fastened to a needle beam or another device104 which is adapted to move up and down in a reciprocating motion asdriven by a main drive 105. The needle board 102 comprises a multitudeof felting or barb needles 103. Barb needles 103 of needle beam ordevice 104 penetrate a mesh 117 and mechanically entangle demineralizedbone fibers 116 into mesh 117. Demineralized bone fibers 106 from inputdevice 107 pass through a batt compression process 108, followed, insome aspects by pressure applied by calendar rolls (not shown) and passbetween a perforated stripper plate 112 to a bed plate 114 where thefilm of demineralized bone fibers 116 is further pressed and/or needlepunched into mesh 117 to form an implantable mesh having demineralizedbone fibers mechanically entangled therein 118. The resulting DBMs arethus reinforced by the mesh into which they are mechanically entangledand can be used as surgical wraps which can be sutured without tearing.In a nonwoven fabric of demineralized bone fibers, the coherent mass ofdemineralized bone fibers is held together by mechanical entanglement ina random web or mat.

FIG. 3 illustrates another embodiment of a process for making animplantable mesh containing demineralized bone fibers mechanicallyentangled therein. FIG. 3 is a simplified schematic of a spun lace orentanglement process 200 of making an implantable mesh 220 containingdemineralized bone fibers 202 mechanically entangled into mesh 218. Inthis process, a bale of demineralized bone fibers either dry 202 or wet204 becomes entangled into mesh 218 by using high velocity jets of wateror air 206 to form the implantable mesh 220. In some aspects, theimplantable mesh 220 can be subjected to a drying process 208 prior toexiting the spun lace process. The water pressure of the water jetinjectors 206 generally increases from the first to the last water jetinjectors. In some embodiments, pressures as high as 2200 psi can beused to direct the water jets onto the web of demineralized bone fibers.

FIG. 4A is a side view of DBM fibers 400. FIG. 4B is a side view ofbioresorbable or permanent mesh 410. FIG. 4C is a side view of theimplantable mesh 420 resulting from mechanically entangling the DBMfibers 400 of FIG. 4A with the mesh 410 of FIG. 4B. The bone fibers 400are mechanically entangled into the mesh along the mesh's longitudinalaxis and the bone fibers can be continuous with the mesh as shown inFIG. 4C. The bone fibers can extend outside of the mesh as shown in FIG.4C. In some embodiments, the bone fiber of the mesh can alternate withthe polymer mesh. Therefore, the mesh can comprise a strand of polymerand then alternate with a strand of fiber, then a strand of polymer, andthen a strand of fiber to have alternating strands that comprise themesh.

In some embodiments, the mesh comprises the same or more polymer thanbone fiber. In some embodiments, the mesh comprises a polymer that makesup from about 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0,14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0,20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0,26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0,32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0,38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0,44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50.0,50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5, 56.0,56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, 60%, 65%, 70%, 75%, 80%, 85%,to about 90% w/w of the mesh.

In some embodiments, the mesh comprises a bone fibers that makes up fromabout 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0,25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0,31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0,37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0,43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0,49.5, 50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0,55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5, to about 60% w/wof the mesh.

In some embodiments the biodegradable fibers of the mesh can beentangled with the demineralized bone fibers to form a plurality oflayers in the implantable mesh (not shown). For example, in otheraspects, bone fibers can be mechanically entangled with non-bone fibersand further mechanically entangled with the biodegradable mesh.

In some embodiments, DBM fibers can be milled, for example, cartridgemilled. The acid extraction process can be conducted so as to leavecollagen, noncollagenous proteins, and growth factors together in asolid fiber. FIG. 5A illustrates an example of DBM sheets and/orshavings 500 containing natural collagen fibers. FIG. 5B illustrates anexample of a mesh 510 that could be used to reinforce the DBM sheetsand/or shavings 500 containing collagen fibers of FIG. 5A. When the DBMsheets/shavings 500 are mechanically entangled with mesh 510,implantable mesh 520 of FIG. 5C obtains. In some embodiments thebiodegradable fibers of the mesh can be entangled with the DBM sheetsand/or shaving containing collagen fibers to form a plurality of layersin the implantable mesh (not shown).

By mechanically entangling the DBM fibers, sheets or shaving into amesh, the DBM fibers are reinforced and can stay together more than from10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% to 100% than if they were not mechanicallyentangled, even after wetting the implantable mesh containing themechanically entangled DBMs.

In some embodiments, the implantable mesh containing the mechanicallyentangled DBMs does not have a carrier or binding agent. Thus, afterentanglement the implantable mesh is 99% or more free of a carrier orbinding agent, yet still holds together. Examples of suitable bindingagents or carrier that optionally can be included after the implantablemesh is formed include, but are not limited to, glycerol, polyglycerol,polyhydroxy compound, for example, such classes of compounds as theacyclic polyhydric alcohols, non-reducing sugars, sugar alcohols, sugaracids, monosaccarides, disaccharides, water-soluble or water dispersibleoligosaccarides, polysaccarides and known derivatives of the foregoing.Specific polyhydroxy compounds include, 1,2-propanediol, glycerol,1,4,-butylene glycol trimethylolethane, trimethylolpropane, erythritol,pentaerythritol, ethylene glycols, diethylene glycol, triethyleneglycol, tetraethylene glycol, propylene glycol, dipropylene glycol;polyoxyethylene-polyoxypropylene copolymer, for example, of the typeknown and commercially available under the trade names Pluronic® andEmkalyx®; polyoxyethylene-polyoxypropylene block copolymer, for example,of the type known and commercially available under the trade namePoloxamer®; alkylphenol-hydroxypolyoxyethylene, for example, of the typeknown and commercially available under the trade name Triton,polyoxyalkylene glycols such as the polyethylene glycols, xylitol,sorbitol, mannitol, dulcitol, arabinose, xylose, ribose, adonitol,arabitol, inositol, fructose, galactose, glucose, mannose, sorbose,sucrose, maltose, lactose, maltitol, lactitol, stachyose, maltopentaose,cyclomaltohexaose, carrageenan, agar, dextran, alginic acid, guar gum,gum tragacanth, locust bean gum, gum arabic, xanthan gum, amylose,mixtures of any of the foregoing.

The carrier or binding agent optionally used may further comprise ahydrogel such as hyaluronic acid, dextran, Pluronic® block copolymers ofpolyethylene oxide and polypropylene, and others. Suitable polyhodroxycompounds include such classes of compounds as acyclic polyhydricalcohols, non-reducing sugars, sugar alcohols, sugar acids,monosaccharides, disaccharides, water-soluble or water dispersibleoligosaccharides, polysaccharides and known derivatives of theforegoing. An example carrier comprises glyceryl monolaurate dissolvedin glycerol or a 4:1 to 1:4 weight mixtures of glycerol and propyleneglycol. Settable materials may be used, and they may set up either insitu, or prior to implantation. Optionally, xenogenic bone powdercarriers also may be treated with proteases such as trypsin. Xenogeniccarriers may be treated with one or more fibril modifying agents toincrease the intraparticle intrusion volume (porosity) and surface area.Useful agents include solvents such as dichloromethane, trichloro-aceticacid, acetonitrile and acids such as trifluoroacetic acid and hydrogenfluoride. The choice of carrier may depend on the desiredcharacteristics of the composition. In some embodiments, a lubricant,such as water, glycerol, or polyethylene glycol may be added.

Compositions and methods are provided for an implantable mesh containingmechanically entangled demineralized bone fibers for hydration with aliquid, the implantable mesh having no carrier disposed in or on it.After the demineralized bone fibers are mechanically entangled into themesh, in some aspects, the resulting implantable mesh can belyophilized. In some embodiments, the demineralized bone fibers arecartridge milled and have a ribbon-like shape and increased surfacearea. In some embodiments, after the demineralized bone fibers arecartridge milled, they can be subjected to process of mechanicalentanglement as discussed above and the resulting implantable mesh canbe subsequently lyophilized.

In some embodiments, the milled and lyophilized mechanically entangleddemineralized bone fibers comprise autograft or allograft bone. In someembodiments, the bone fibers have a diameter from about 100 μm to about2 mm. In some embodiments, the bone fibers have a length from about 0.5mm to about 50 mm. In some embodiments, the bone fibers have an averagelength from about 0.5 cm to about 10 cm. In some embodiments, the fibershave an aspect ratio of from about 50:1 to about 1000:1, from about 50:1to about 950:1, from about 50:1 to about 750:1, from about 50:1 to about500:1, from about 50:1 to about 250:1, from about 50:1 to about 100:1,from about 10:1 to about 50:1, or from about 5:1 to about 10:1. In someembodiments, the liquid for hydration of the fibers comprises blood,water, saline or a combination thereof. In some embodiments, the liquidfor hydration of the fibers is mixed with the implantable mesh havingmilled and demineralized bone fibers mechanically entangled therein toform moldable lyophilized demineralized bone fiber implantable mesh.

In some embodiments, the milled and lyophilized demineralized bonefibers that are mechanically entangled into the mesh do not contain acarrier. In some aspects, the demineralized bone fibers comprisecartridge milled fibers having a curled portion. In some embodiments,the implantable mesh of milled and lyophilized demineralized bone fiberscomprises autograft or allograft bone. In some embodiments, the bonefibers have a diameter from about 100 μm to about 2 mm. In someembodiments, the bone fibers have a length from about 0.5 mm to about 50mm. In some embodiments, the bone fibers have an average length fromabout 0.5 cm to about 10 cm. In some embodiments, the fibers have anaspect ratio of from about 50:1 to about 1000:1, from about 50:1 toabout 950:1, from about 50:1 to about 750:1, from about 50:1 to about500:1, from about 50:1 to about 250:1, from about 50:1 to about 100:1,from about 10:1 to about 50:1, or from about 5:1 to about 10:1. In someembodiments, the liquid for hydration of the fibers comprisesphysiologically acceptable water, physiological saline, sodium chloride,dextrose, Lactated Ringer's solution, phosphate buffered saline (PBS),blood, bone marrow aspirate, bone marrow fractions or a combinationthereof in an amount sufficient to render the implantable osteogenicmaterial moldable. In some embodiments, the liquid is mixed with thelyophilized implantable mesh containing mechanically entangleddemineralized bone fibers to form moldable lyophilized demineralizedbone fiber.

In some embodiments, the implantable mesh comprises cortical bone,cancellous bone, cortico-cancellous bone, or mixtures thereof. In someembodiments, the bone material in the implantable mesh is obtained fromautogenous bone, allogenic bone, xenogenic bone, or mixtures thereof. Insome embodiments, the implantable mesh is lyophilized and shaped. Insome embodiments, the shape of the lyophilized implantable mesh is acube, square, triangle, rectangle, circle, disc or cylinder shape. Insome embodiments, the shape of the implantable mesh having mechanicallyentangled demineralized bone fibers therein is disc or cylinder shapedand the disc or cylinder has a reservoir configured to contact a liquid.Compositions and methods are provided for an implantable bone graftcomprising fibers obtained from allograft bone, the fibers comprisinghooking portions configured to entangle with a biodegradable orpermanent mesh to form an implantable mesh, wherein the composition doesnot include a binding agent.

Typically, when bone is processed into particles or fibers, it isstatically charged and not coherent or adherent. The processed bone isnormally contained within an external structure (i.e., a bag orcovering) or mixed with a carrier or binding agent to provide a cohesivestructure. When implanted, this external structure or carrier must beremoved by the patient's body, potentially impacting the osteoinductivepotential of the graft.

In some embodiments, the implantable mesh described in this applicationcontains demineralized bone fibers which are mechanically entangled byneedle punching, entanglement pressure, water or air jet or sonicationto form reinforced DBMs having enhanced cohesion between fibers withouta requirement for additional containment, carrier or binding agents.

In some embodiments, the curled bone fibers can be further subjected tomechanical entanglement as discussed above, so that the resultingimplantable mesh is like felt in consistency and can be easily shapedinto desired shapes. Further, in some aspects, the milled and/or curledfiber shape is altered during the drying process, which leads tophysical entanglement and surface to surface interactions betweenadjacent fibers. In some embodiments, the milled fibers are subjected tothe mechanical entanglement processes discussed above, namely needlepunching or entanglement. The entanglement/interaction of the fibers isresponsible for the cohesiveness of the final implantable mesh.

The compositions of the present disclosure results are utilized in aneffective bone grafting product. The bone graft material is resorbedand/or remodeled and replaced by host bone during the healing process.In some embodiments, the bone material disclosed herein includesadditional additives, such as synthetic ceramics and/or bioerodiblepolymers, which produce high concentrations of calcium, phosphate andsilicon ions that act as a nidus for de-novo bone formation, asdiscussed herein. As the bioerodible polymer degrades faster than theceramic, more and more osteoinductive DBM particles are exposed. Theslower resorbing ceramic may act as a solid surface for stem cells andosteoblasts to attach to and begin laying down new bone.

The implantable mesh of this disclosure has good flexibility and iscompression resistant. It is also osteoinductive with the demineralizedbone matrix retaining activity. These properties make an excellent bonegraft substitute in that it may not break, crack, or deform whenimplanted in the body.

The implantable mesh may include among its mechanically entangled DBMs acombination of fibers of bone matrix from allograft bone and fibers ofnon-allograft bone material. The fibers of the non-allograft bonematerial comprise non-fibrous demineralized bone matrix particlesembedded within or dispersed on the fibers of the non-allograft bonematerial. The ratio of fibers of demineralized bone matrix fromallograft material to fibers of non-allograft material ranges from about20:80 to about 70:30. In one embodiment, the ratio of fibers fromallograft material to fibers of non-allograft material ranges from about40:60 to about 60:40. In one embodiment, the ratio of fibers ofdemineralized bone matrix from allograft material to fibers ofnon-allograft material is about 50:50.

In some embodiments, the demineralized bone material includes particlesthat are non-fibrous. In some embodiments, the particles are powders,microspheres, sponges, pastes, gels, and/or granules. In one embodiment,the particles are powders.

In some embodiments, the demineralized bone material fibers comprisefrom about 1 to about 70 micrometers or from about 125 to about 250micrometers. In some embodiments, the demineralized bone material fiberscomprise about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248 and/or 250 micrometers. In some embodiments, the bonefibers include a length from about 100 micrometers to about 2 mm. Insome embodiments, the bone fibers have a length from about 0.5 cm toabout 10 cm, about 1 cm to about 8 cm, about 3 cm to about 5 cm, about0.5 mm to about 50 mm, about 1.0 mm to about 25 mm, or about 5 mm toabout 10 mm. The fibers include a diameter of about 100 micrometers toabout 2 mm.

The fibers are milled in such a way as to provide increased surface areain a compact shape and size. In some embodiments, the fibers include acurled shape such that diameter of the curled fibers is between about 50micrometers and about 3 mm, and the diameter of the fibers in aflattened configuration is about 125 micrometers to about 5 mm. In someembodiments, the fibers include a curled shape such that diameter of thecurled fibers is between about 100 micrometers and about 1 mm, and thediameter of the fibers in a flattened configuration is about 250micrometers to about 2 mm.

In various embodiments, the fibers have an aspect ratio of length towidth from about 50:1 to about 1000:1, from about 50:1 to about 950:1,from about 50:1 to about 750:1, from about 50:1 to about 500:1, fromabout 50:1 to about 250:1, from about 50:1 to about 100:1, from about10:1 to about 50:1, or from about 5:1 to about 10:1. In otherembodiments, the fibers have an aspect ratio of length to width of about4:1, 17:1, or 23:1.

The composition has very low immunogenicity and good compatibility.

DBM fibers for use in the present disclosure can be obtainedcommercially or can be prepared by known techniques. In general,advantageous, osteoinductive DBM materials can be prepared bydecalcification of cortical and/or cancellous bone fibers, often by acidextraction. The fibers can be milled, for example, cartridge milled. Theacid extraction process can be conducted so as to leave collagen,noncollagenous proteins, and growth factors together in a solid fiber.Methods for preparing bioactive demineralized bone are described in U.S.Pat. Nos. 5,073,373; 5,484,601; and 5,284,655, as examples. DBM productsare also available commercially, including for instance, from sourcessuch as Regeneration Technologies, Inc. (Alachua, Fla.), The AmericanRed Cross (Arlington, Va.), and others. Bone fibers that are solelyosteoconductive can be prepared using similar techniques that have beenmodified or supplemented to remove or inactivate (e.g. by crosslinkingor otherwise denaturing) components in the bone matrix responsible forosteoinductivity. Osteoinductive and/or osteoconductive DBM materialsused in the present disclosure can be derived from human donor tissue,especially in regard to implant devices intended for use in humansubjects.

In regard to the fiber content of the implantable mesh on a dry weightbasis, the bone fiber material can constitute about 5% to about 100% ofthe compositions, about 20% to about 80%, or about 25% to about 75% byweight.

In some embodiments, the bone fibers of allograft bone have an averagelength to average thickness ratio or aspect ratio of the fibers fromabout 50:1 to about 1000:1. In overall appearance the bone fibers can bein the form of ribbons, threads, narrow strips, and/or thin sheets. Theelongated bone fibers can be substantially linear in appearance or theycan be coiled to resemble springs. In some embodiments, the bone fibershave linear portions and coiled portions. In some embodiments, the bonefibers are of irregular shapes including, for example, linear,serpentine and/or curved shapes. In some embodiments, the fibers can becurled at the edges to have a substantially hemicircular cross-sections.In some embodiments, the fibers may be entirely or partially helical,circumvoluted or in the shape of a corkscrew. The elongated bone fiberscan be demineralized however some of the original mineral content may beretained when desirable for a particular embodiment. The bone graftfiber may further comprise mineralized bone material.

The bone fiber sizes and shapes may be created in a number of ways, forexample, through cartridge milling. One such example of a suitablecartridge mill is the Osteobiologic Milling Machine, as described inU.S. Patent Publication No. 2012/0160945, assigned to Warsaw Orthopedic,Inc. and is hereby incorporated by reference in its entirety. However,it is contemplated that the bone fibers may be alternatively milledusing vices, cutters, rollers, rotating rasps or reciprocating blademills.

Non-Bone Material Additives p In some embodiments, the bone fibers maybe combined with non-bone material additives after demineralizationand/or lyophilization and before implantation. For example, the bonefibers may be combined with a bioerodible polymer. The bioerodiblepolymer exhibits dissolution when placed in a mammalian body and may behydrophilic (e.g., collagen, hyaluronic acid, polyethylene glycol).Synthetic polymers are suitable according to the present disclosure, asthey are biocompatible and available in a range of copolymer ratios tocontrol their degradation.

In some embodiments, hydrophobic polymers (e.g. poly(lactide-co-glycolyde), polyanhydrides) may be used. Alternatively, acombination of hydrophilic and hydrophobic polymers may be used in thebone graft composition of the disclosure.

Exemplary materials may include biopolymers and synthetic polymers suchas human skin, human hair, bone, collagen, fat, thin crosslinked sheetscontaining fibers and/or fibers and chips, polyethylene glycol (PEG),chitosan, alginate sheets, cellulose sheets, hyaluronic acid sheet, aswell as copolymer blends of poly (lactide-co-glycolide) PLGA.

Useful bioerodible polymers may have a molecular weight of from about1,000 to about 30,000 Daltons (Da). In various embodiments, the polymermay have a molecular weight of from about 2,000 to about 10,000 Da. Insome embodiments, the polymer may have a molecular weight of from about2,000 to 4,000 Da or from about 3,000 to 4,000 Da. In some embodiments,the bioerodible polymer may have a molecular weight of 1,000, 2,000,3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000,22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000 or about30,000 Da.

In some embodiments, the bioerodible polymer can be collagen. Collagenhas excellent histocompatibility without antibody formation or graftrejection. Any suitable collagen material may be used, including knowncollagen materials, or collagen materials as disclosed in U.S. patentapplication Ser. No. 12/030,181, filed Feb. 12, 2008, herebyincorporated by reference in its entirety.

Various collagen materials can be used, alone or in combination withother materials present in the implantable mesh described in thisdisclosure. In some embodiments, the implantable mesh containingmechanically entangled demineralized bone fibers comprises abiodegradable polymer, such as, for example, collagen. In someembodiments, the biodegradable polymer is crosslinked. Exemplarycollagens include human or non-human (bovine, ovine, piscine, and/orporcine), as well as recombinant collagen or combinations thereof.Examples of suitable collagen include, but are not limited to, humancollagen type I, human collagen type II, human collagen type III, humancollagen type IV, human collagen type V, human collagen type VI, humancollagen type VII, human collagen type VIII, human collagen type IX,human collagen type X, human collagen type XI, human collagen type XII,human collagen type XIII, human collagen type XIV, human collagen typeXV, human collagen type XVI, human collagen type XVII, human collagentype XVIII, human collagen type XIX, human collage type XX, humancollagen type XXI, human collagen type XXII, human collagen type XXIII,human collagen type XXIV, human collagen type XXV, human collagen typeXXVI, human collagen type XXVII, and human collagen type XXVIII, orcombinations thereof. Collagen further may comprise hetero- andhomo-trimers of any of the above-recited collagen types. In someembodiments, the collagen comprises hetero- or homo-trimers of humancollagen type I, human collagen type II, human collagen type III, orcombinations thereof. In various embodiments, the collagen may becrosslinked.

Insoluble collagen material for use in the disclosure can be derivedfrom natural tissue sources, (e.g. xenogenic, allogenic, or autogenicrelative to the recipient human or other patient) or recombinantlyprepared. Collagens can be subclassified into several different typesdepending upon their amino acid sequence, carbohydrate content and thepresence or absence of disulfide crosslinks. Types I and III collagenare two of the most common subtypes of collagen and may be used in thepresent disclosure. Type I collagen is present in skin, tendon and bone,whereas Type III collagen is found primarily in skin. The collagen usedin compositions of the disclosure can be obtained from skin, bone,tendon, or cartilage and purified by methods well known in the art andindustry. Alternatively, the collagen can be purchased from commercialsources.

The collagen can be atelopeptide collagen and/or telopeptide collagen.Still further, either or both of non-fibrillar and fibrillar collagencan be used. Non-fibrillar collagen is collagen that has beensolubilized and has not been reconstituted into its native fibrillarform.

Suitable collagen products are available commercially, including forexample from DSM Biomedical (Exton, Pa.), which manufactures a fibrouscollagen known as Semed F, from bovine tendon or hides. Collagenmaterials derived from bovine hide are also manufactured by Integra LifeScience Holding Corporation (Plainsboro, N.J.). Naturally-derived orrecombinant human collagen materials are also suitable for use in thedisclosure. Illustratively, recombinant human collagen products areavailable from Fibrogen, Inc. (San Francisco, Calif.).

In some embodiments, the fibers can be combined with synthetic ceramicsthat are effective to provide a scaffold for bone growth and which arecompletely bioresorbable and biocompatible. The synthetic ceramicsshould provide high local concentrations of calcium, phosphate andsilicon ions that act as a nidus for de-novo bone formation. The use ofsuch a resorbable ceramics provides many advantages over alternativeconventional materials. For instance, it eliminates the need forpost-therapy surgery for removal and degrades in the human body tobiocompatible, bioresorbable products.

In some embodiments, the synthetic ceramics disclosed herein may beselected from one or more materials comprising calcium phosphateceramics or silicon ceramics. Biological glasses such ascalcium-silicate-based bioglass, silicon calcium phosphate, tricalciumphosphate (TCP), biphasic calcium phosphate, calcium sulfate,hydroxyapatite, coralline hydroxyapatite, silicon carbide, siliconnitride (Si₃N₄), and biocompatible ceramics may be used. In someembodiments, the ceramic is tri-calcium phosphate or biphasic calciumphosphate and silicon ceramics. In some embodiments, the ceramic istricalcium phosphate.

In some embodiments, the ceramics are a combination of a calciumphosphate ceramic and silicon ceramic. In some embodiments, the calciumphosphate ceramic is resorbable biphasic calcium phosphate (BCP) orresorbable tri-calcium phosphate (TCP), in some aspects, resorbable TCP.

Biphasic calcium phosphate can have a tricalciumphosphate:hydroxyapatite weight ratio of about 50:50 to about 95:5,about 70:30 to about 95:5, about 80:20 to about 90:10, or about 85:15.The mineral material can be a granular particulate having an averageparticle diameter between about 0.2 and 5.0 mm, between about 0.4 and3.0 mm, or between about 0.4 and 2.0 mm.

The ceramics of the disclosure may also be oxide ceramics such asalumina (Al₂O₃) or zirconia (ZrO₂) or composite combinations of oxidesand non-oxides such as silicon nitride).

In some embodiments, the composition containing the fibers may alsocontain other beneficial substances including, for example,preservatives, co-solvents, suspending agents, viscosity enhancingagents, ionic strength and osmolality adjusters and/or other excipients.Suitable buffering agents can also be used an include, but are notlimited to, alkaline earth metal carbonates, phosphates, bicarbonates,citrates, borates, acetates, succinates, or others.Illustrative-specific buffering agents include for instance sodiumphosphate, sodium citrate, sodium borate, sodium acetate, sodiumbicarbonate, sodium carbonate, and sodium tromethanine (TRIS).

In some embodiments, the implantable mesh containing mechanicallyentangled demineralized bone fibers may be mixed with a porogen materialwhich is later removed during manufacturing to enhance porosity of thedried implantable mesh. Suitable porogen materials may be made of anybiocompatible, biodegradable substance that can be formed into aparticle and that is capable of at least substantially retaining itsshape during the manufacturing of the implant, but is later removed ordegrades or dissolves when placed in contact with an aqueous solution,or other liquid. The porogens, in some embodiments, may be inorganic ororganic, for example, they may be made from gelatin, an organic polymer(e.g., polyvinyl alcohol), polyurethanes, polyorthoesters, PLA, PGA, andPLGA copolymers, a saccharide, a calcium salt, sodium chloride, calciumphosphate or mixtures thereof. Porogen particles may be about 100 toabout 500 microns.

In one embodiment, all porogen particles of a given morphology can haveat least one average axial, transverse, or lateral dimension that isabout 100 to about 500 microns. In some embodiments, all porogenparticles used can independently have at least one axial, transverse, orlateral dimension that is about 100 to about 500 microns. In someembodiments, all porogen particles used can collectively have at leastone average axial, transverse, or lateral dimension that is about 100 toabout 500 microns. In some embodiments, at least one dimension of theporogen particles can be about 100 microns or more, or about 120 micronsor more, or about 140 microns or more. In some embodiments, at least onedimension of the porogen particles can be about 500 microns or less,about 425 microns or less, about 350 microns or less, about 300 micronsor less, or about 250 microns or less. In some embodiments, the porogenparticles can have at least one dimension that is about 120 to about 400microns.

In some embodiments the implantable mesh containing demineralized bonefibers mechanically entangled therein could contain single or multipleconcentrations of size controlled fibers to affect its consistency andaffect the handling of the implantable mesh after hydration.

In some instances multiple implantable meshes might be packaged togetherto improve their handling prior to and after hydration. In otherinstances the implantable meshes may be hydrated with a polar ornon-polar solutions and/or salt solutions prior to drying to enhancelater rehydration.

One of more biologically active ingredients may be added to theresulting composition (for example, lyophilized bone fibers). Theseactive ingredients may or may not be related to the bone repaircapabilities of the composition. Suitable active ingredients hemostaticagents, bone morphogenic proteins (BMPs), genes, growth differentiationfactors (GDFs), or other non-collagenic proteins such as TGF-β, PDGF,ostropontin, osteonectin, cytokines, and the like.

In one embodiment, the implantable mesh containing mechanicallyentangled demineralized bone fibers therein can include at least oneBMP, which are a class of proteins thought to have osteoinductive orgrowth-promoting activities on endogenous bone tissue, or function aspro-collagen precursors. Known members of the BMP family include, butare not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7,BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, BMP-16, BMP-17,BMP-18 as well as polynucleotides or polypeptides thereof, as well asmature polypeptides or polynucleotides encoding the same.

BMPs utilized as osteoinductive agents comprise one or more of BMP-1;BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11;BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as anycombination of one or more of these BMPs, including full length BMPs orfragments thereof, or combinations thereof, either as polypeptides orpolynucleotides encoding the polypeptide fragments of all of the recitedBMPs. The isolated BMP osteoinductive agents may be administered aspolynucleotides, polypeptides, full length protein or combinationsthereof.

In another embodiment, the implantable mesh containing demineralizedbone fibers mechanically entangled therein can also include one or moreGrowth Differentiation Factors (“GDFs”) disposed in it. Known GDFsinclude, but are not limited to, GDF-1, GDF-2, GDF-3, GDF-7, GDF-10,GDF-11, and GDF-15. For example, GDFs useful as isolated osteoinductiveagents include, but are not limited to, the following GDFs: GDF-1polynucleotides or polypeptides corresponding to GenBank AccessionNumbers M62302, AAA58501, and AAB94786, as well as mature GDF-1polypeptides or polynucleotides encoding the same. GDF-2 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers BC069643,BC074921, Q9UK05, AAH69643, or AAH74921, as well as mature GDF-2polypeptides or polynucleotides encoding the same. GDF-3 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AF263538,BC030959, AAF91389, AAQ89234, or Q9NR23, as well as mature GDF-3polypeptides or polynucleotides encoding the same. GDF-7 polynucleotidesor polypeptides corresponding to GenBank Accession Numbers AB158468,AF522369, AAP97720, or Q7Z4P5, as well as mature GDF-7 polypeptides orpolynucleotides encoding the same. GDF-10 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers BC028237 orAAH28237, as well as mature GDF-10 polypeptides or polynucleotidesencoding the same.

The implantable mesh containing demineralized bone fibers mechanicallyentangled therein can also include: GDF-11 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers AF100907,NP005802 or 095390, as well as mature GDF-11 polypeptides orpolynucleotides encoding the same; GDF-15 polynucleotides orpolypeptides corresponding to GenBank Accession Numbers BC008962,BC000529, AAH00529, or NP004855, as well as mature GDF-15 polypeptidesor polynucleotides encoding the same.

In some embodiments, the implantable mesh having demineralized bonefibers mechanically entangled therein contains other bioactive agentswhich can be delivered with it. In certain embodiments, the bioactiveagent is a drug. These bioactive agents may include, for example,antimicrobials, antibiotics, antimyobacterial, antifungals, antivirals,antineoplastic agents, antitumor agents, agents affecting the immuneresponse, blood calcium regulators, agents useful in glucose regulation,anticoagulants, antithrombotics, antihyperlipidemic agents, cardiacdrugs, thyromimetic and antithyroid drugs, adrenergics, antihypertensiveagents, cholinergic, anticholinergics, antispasmodics, antiulcer agents,skeletal and smooth muscle relaxants, prostaglandins, general inhibitorsof the allergic response, antihistamines, local anesthetics, analgesics,narcotic antagonists, antitussives, sedative-hypnotic agents,anticonvulsants, antipsychotics, anti-anxiety agents, antidepressantagents, anorexigenics, non-steroidal anti-inflammatory agents, steroidalanti-inflammatory agents, antioxidants, vaso-active agents, bone-activeagents, osteogenic factors, antiarthritics, and diagnostic agents. Amore complete listing of bioactive agents and specific drugs suitablefor use in the present disclosure may be found in “The Merck Index: AnEncyclopedia of Chemicals, Drugs, and Biologicals,” Edited by SusanBudavari, et al.; and the United States Pharmacopoeia/National FormularyXXXVII/XXXII, published by the United States Pharmacopeial Convention,Inc., Rockville, Md., 2013, each of which is incorporated herein byreference.

Bioactive agents may also be provided by incorporation into theimplantable mesh having mechanically entangled demineralized bone fiberstherein. Bioactive agents such as those described herein can beincorporated homogeneously or regionally into the implant material bysimple admixture or otherwise. Further, they may be incorporated aloneor in conjunction with another carrier form or medium such asmicrospheres or another microparticulate formulation. Suitabletechniques for forming microparticles are well known in the art, and canbe used to entrain or encapsulate bioactive agents, whereafter themicroparticles can be dispersed within the bone graft composite upon orafter its preparation.

It will be appreciated that the amount of additive used will varydepending upon the type of additive, the specific activity of theparticular additive preparation employed, and the intended use of thecomposition. The desired amount is readily determinable by the user.

Any of a variety of medically and/or surgically useful substances can beincorporated in, or associated with, the allograft bone material eitherbefore, during, or after preparation of the implantable mesh containingmechanically entangled demineralized bone fibers. Thus, for example,when the non-allograft bone material is used, one or more of suchsubstances may be introduced into the bone fibers, for example, bysoaking or immersing these bone fibers in a solution or dispersion ofthe desired substance(s).

In some embodiments, the implantable mesh containing biodegradablefibers mechanically entangled therein can be lyophilized with one ormore growth factors (e.g., BMP, GDF), drugs so that it can be releasedfrom the implantable mesh in a sustained release manner.

Bone Fiber Shapes

The present disclosure also provides methods for shaping the implantablemesh containing mechanically entangled demineralized bone fiberstherein. The fibers utilized in the implantable mesh, in some aspectscan be milled from bone shafts using any appropriate apparatus, such asa cartridge mill. The fibers are milled to include curled shapes havingfrayed portions and/or hooked portions to facilitate mechanicalentanglement of the fibers. The shape of the allograft may be tailoredto fit the site at which it is to be situated. For example, it may be inthe shape of a morsel, a plug, a pin, a peg, a cylinder, a block, awedge, ring, or a sheet. In some embodiments, the implantable mesh maybe shaped as a wrap for wrapping around a bone defect and to containother allograft or synthetic material into which a surgeon or anothermedical practitioner can safely place sutures without unraveling.

In one embodiment, the method comprises placing the implantable meshhaving mechanically entangled demineralized bone fibers into a moldprior to demineralization and/or lyophilization. The bone fibers in theimplantable mesh are then demineralized, sterilized and/or lyophilizedto create a shaped mesh containing mechanically entangled demineralizedbone fibers. The bone fibers mechanically entangled into the implantablemesh can be placed into a mold and then subjected to demineralizationand/or lyophilization to make the desired shape or the bone fibers canbe demineralized, mechanically entangled into the implantable meshand/or lyophilized and then shaped by stamping or punching the desiredshape. The demineralization and lyophilization steps alter the shape ofthe bone fibers to facilitate mechanical entanglement, as discussedherein. The bone fibers mechanically entangled into the implantable meshdo not require the use of a binding agent or carrier to form theimplantable mesh.

In some embodiments, the demineralized bone fibers mechanicallyentangled into a mesh can be placed into molds and shaped to form a in arange of predetermined shapes and sizes according to the needs of amedical procedure. In some embodiments, the allograft may be made byinjection molding, compression molding, die pressing, slip casting,laser cutting, water-jet machining, sand casting, shell mold casting,lost tissue scaffold casting, plaster-mold casting, vacuum casting,permanent-mold casting, slush casting, pressure casting, die casting,centrifugal casting, squeeze casting, rolling, forging, swaging,extrusion, shearing, spinning, or combinations thereof. For example, theimplantable mesh may be rectangular, pyramidal, triangular, pentagonal,or other polygonal or irregular prismatic shapes.

Demineralization

After the bone is obtained from the donor it can be demineralized beforeor after it is formed into a fiber. In some embodiments, after the boneis obtained from the donor and milled into a fiber, it is processed,namely, cleaned, disinfected, and defatted using methods well known inthe art. The entire bone can then be demineralized or, if desired, thebone can just be sectioned before demineralization. The entire bone orone or more of its sections is then subjected to demineralization inorder to reduce the inorganic content to a low level, e.g., to containless than about 10% by weight, in some aspects, less than about 5% byweight and in other aspects, less than about 1% by weight, residualcalcium.

DBM may be prepared in any suitable manner. In one embodiment, the DBMis prepared through the acid extraction of minerals from bone. Itincludes the collagen matrix of the bone together with acid insolubleproteins including bone morphogenic proteins (BMPs) and other growthfactors. It can be formulated for use as granules, gels, sponge materialor putty and can be freeze-dried for storage. Sterilization proceduresused to protect from disease transmission may reduce the activity ofbeneficial growth factors in the DBM. DBM provides an initialosteoconductive matrix and exhibits a degree of osteoinductivepotential, inducing the infiltration and differentiation ofosteoprogenitor cells from the surrounding tissues. As noted, inembodiments of bone particles taken from cortical long bones, theosteoinductive potential of the bone particles when demineralized mayvary based on the source of the bone particles, whether from theperiosteal layer, the middle layer, or the endosteal layer.

DBM preparations have been used for many years in orthopedic medicine topromote the formation of bone. For example, DBM has found use in therepair of fractures, in the fusion of vertebrae, in joint replacementsurgery, and in treating bone destruction due to underlying disease suchas rheumatoid arthritis. DBM is thought to promote bone formation invivo by osteoconductive and osteoinductive processes. The osteoinductiveeffect of implanted DBM compositions is thought to result from thepresence of active growth factors present on the isolated collagen-basedmatrix. These factors include members of the TGF-β, IGF, and BMP proteinfamilies. Particular examples of osteoinductive factors include TGF-β,IGF-1, IGF-2, BMP-2, BMP-7, parathyroid hormone (PTH), and angiogenicfactors. Other osteoinductive factors such as osteocalcin andosteopontin are also likely to be present in DBM preparations as well.There are also likely to be other unnamed or undiscovered osteoinductivefactors present in DBM.

In one demineralization procedure, the implantable mesh can be subjectedto an acid demineralization step followed by a defatting/disinfectingstep. The implantable mesh containing the bone fibers mechanicallyentangled therein is immersed in acid to effect demineralization. Acidsthat can be employed in this step include inorganic acids such ashydrochloric acid and as well as organic acids such as formic acid,acetic acid, peracetic acid, citric acid and/or propionic acid. Thedepth of demineralization into the bone surface can be controlled byadjusting the treatment time, temperature of the demineralizingsolution, concentration of the demineralizing solution, and agitationintensity during treatment. Thus, in various embodiments, the DBM may befully demineralized, partially demineralized, or surface demineralized.

The demineralized bone is rinsed with sterile water and/or bufferedsolution(s) to remove residual amounts of acid and thereby raise the pH.A suitable defatting/disinfectant solution is an aqueous solution ofethanol, the ethanol being a good solvent for lipids and the water beinga good hydrophilic carrier to enable the solution to penetrate moredeeply into the bone particles. The aqueous ethanol solution alsodisinfects the bone by killing vegetative microorganisms and viruses.Ordinarily, at least about 10 to 40 percent by weight of water (i.e.,about 60 to 90 weight percent of defatting agent such as alcohol) ispresent in the defatting disinfecting solution to produce optimal lipidremoval and disinfection within a given period of time. A suitableconcentration range of the defatting solution is from about 60 to about85 weight percent alcohol, or about 70 weight percent alcohol.

In some embodiments, the demineralized bone may be further treated toeffect properties of the bone. For example, the DBM may be treated todisrupt the collagen structure of the DBM. Such treatment may comprisecollagenase treatment, heat treatment, mechanical treatment, or other.Reference is made to U.S. Provisional Patent Applications 60/944,408;60/944,417; and 60/957,614, herein incorporated by reference, forfurther treatment options.

Lyophilization

The bone fibers are lyophilized either in a mold for a desired shape orout of a mold, where it can be shaped (e.g., stamped, punched, cut). Forexample, the bottle containing bone and conserving agent is initiallyfrozen to −76° C. with the bone and conserving agent later beingsubjected to a vacuum of less than 100 millitorr while the temperatureis maintained at or below −35° C. The end point of the lyophilizationprocedure is the determination of residual moisture of approximately 5%.Once the bone has been lyophilized, it is stored in sealed,vacuum-contained, bottles prior to its reconstitution and use.

In some embodiments, the demineralization and lyophilization steps alterthe shape of the fibers to facilitate mechanical entanglement. Tofacilitate on-site preparation and/or usage of the implantable mesh, thedemineralized fibrous bone elements and non-fibrous bone elements, insome embodiments, in lyophilized or frozen form, and fluid carrier (thelatter containing one or more optional ingredients such as thoseidentified above) can be stored in separate packages or containers understerile conditions and brought together in intimate admixture at themoment of use for immediate application to an osseous defect siteemploying any suitable means such as spatula, forceps, syringe, tampingdevice, and the like. Alternatively, the implantable mesh can beprepared well in advance and stored under sterile conditions untilrequired for use. When the implantable mesh is prepared well in advanceit is lyophilized, in some aspects, prior to packaging for storage. Insome embodiments, the implantable mesh described herein can be combinedwith autograft bone marrow aspirate, autograft bone, preparations ofselected autograft cells, autograft cells containing genes encoding bonepromoting action prior to being placed in a defect site. In variousembodiments, the implantable mesh is packaged already mixed and readyfor use in a suitable container, such as for example, resealablenon-toxic bottle, a mesh bag or pouch, or is provided as a kit which canbe prepared at a surgeon's direction when needed.

Hydration of Implant

In some embodiments, the implantable mesh containing demineralized bonefibers mechanically entangled therein is hydrated with physiologicallyacceptable water, physiological saline, sodium chloride, dextrose,Lactated Ringer's solution, phosphate buffered saline, blood, bonemarrow aspirate, bone marrow fractions or a combination thereof in anamount sufficient to render the implantable osteogenic materialmoldable. Once hydrated, the implantable mesh is placed into a surgicalsite at a location determined by a medical practitioner. The fibers inthe implantable mesh maintain their coherency and mechanicalinteractions such that the mesh does not require a binding agent orcarrier when placed in situ. In some embodiments, the fibers of theimplantable mesh are hydrophobic and internal or external hydrationchannels facilitate hydration of the implantable mesh.

In some embodiments, the implantable mesh may be hydrated with PBS orother physiologically acceptable fluid, and provided for use in ahydrated form. The implantable mesh may be placed at a surgical sitedirectly and subsequently hydrated. In some embodiments, the implantablemesh can be wrapped around a bone defect or can help contain otherallograft or synthetic material.

A physiologically acceptable liquid, in some embodiments containingwater, may be added to the implantable mesh prior to placement into thesite or wrapped around the bone defect. Such physiologically acceptableliquids include those discussed above, including physiological saline ora blood product. Blood products include whole blood and blood fractionssuch as platelet rich plasma and platelet poor plasma.

In some embodiments, the implantable mesh is hydrated with aphysiologically acceptable liquid and biocompatible carrier.Non-limiting examples of physiologically acceptable liquids includesaline, phosphate buffered saline (PBS), hyaluronic acid, celluloseethers (such as carboxymethyl cellulose), collagen, gelatin, autoclavedbone powder, osteoconductive carriers, whole blood, blood fractions,bone marrow aspirate, concentrated bone marrow aspirate, and mixturesthereof. Non-limiting examples of blood fractions include serum, plasma,platelet-rich plasma, concentrated platelet-rich plasma, platelet-poorplasma, and concentrated platelet poor plasma. After hydrating, theimplantable mesh can be molded into a predetermined shape oradministered to or wrapped around a bone defect and manipulated toconform to the bone defect in such a manner that will promote healing.For example, the composition may be hydrated with about 2 ml of salineblood per 2.5 g of combined DBM and periosteal powder.

Methods of Treatment

Illustrative bone repair sites that can be treated with implantable meshof the disclosure include, for instance, those resulting from injury,defects brought about during the course of surgery, infection,malignancy or developmental malformation. The implantable meshcontaining demineralized bone fibers mechanically entangled therein canbe used in a wide variety of orthopedic, periodontal, neurosurgical andoral and maxillofacial surgical procedures including, but not limited tothe repair of simple and compound fractures and non-unions; external andinternal fixations; joint reconstructions such as arthrodesis; generalarthroplasty; cup arthroplasty of the hip; femoral and humeral headreplacement; femoral head surface replacement and total jointreplacement; repairs of the vertebral column including spinal fusion andinternal fixation; tumor surgery, e.g., deficit filing; discectomy;laminectomy; excision of spinal cord tumors; anterior cervical andthoracic operations; repairs of spinal injuries; scoliosis, lordosis andkyphosis treatments; intermaxillary fixation of fractures; mentoplasty;temporomandibular joint replacement; alveolar ridge augmentation andreconstruction; inlay osteoimplants; implant placement and revision;sinus lifts; cosmetic enhancement; etc. Specific bones which can berepaired or replaced with the implantable mesh include, but are notlimited to the ethmoid; frontal; nasal; occipital; parietal; temporal;mandible; maxilla; zygomatic; cervical vertebra; thoracic vertebra;lumbar vertebra; sacrum; rib; sternum; clavicle; scapula; humerus;radius; ulna; carpal bones; metacarpal bones; phalanges; ilium; ischium;pubis; femur; tibia; fibula; patella; calcaneus; tarsal and metatarsalbones.

In accordance with certain aspects of the disclosure, the implantablemesh of the disclosure can be used as bone void fillers, or can beincorporated in, on or around load bearing implants such as spinalimplants, hip implants (e.g. in or around implant stems and/or behindacetabular cups), knee implants (e.g. in or around stems). In someembodiments, the implantable mesh of the disclosure can be incorporatedin, on or around a load-bearing spinal implant device having acompressive strength of at least about 10000 N, such as a fusion cage,PEEK implants, dowel, or other device potentially having a pocket,chamber or other cavity for containing an osteoinductive composition,and used in a spinal fusion such as an interbody fusion. Oneillustrative such use is in conjunction with a load-bearing interbodyspinal spacer to achieve interbody fusion. In these applications, theimplantable mesh can be placed in and/or wrapped around the spacer tofacilitate the fusion.

Methods for preparing DBM are well known in the art as described, forexample, in U.S. Pat. Nos. 5,314,476; 5,507,813; 5,073,373; and5,405,390, each incorporated herein by reference. Methods for preparingceramic powders of calcium phosphate and/or hydroxyapatite aredescribed, e.g., in U.S. Pat. Nos. 4,202,055 and 4,713,076, eachincorporated herein by reference.

In some embodiments, the method comprises obtaining the fibers byshaving, milling, or pressing the sheet or block under asepticconditions. The shape of the fibers can be optimized for inducing newbone formation and handling properties via the network of fibers.

In a still further aspect, the present disclosure provides a method ofaccelerating bone formation at an implantable tissue regenerationscaffold. In a still further aspect, the present disclosure provides amethod of regenerating bone in a patient in need thereof, comprisingimplanting the patient with the implantable mesh.

In a still further aspect, the present disclosure provides a method oftreating a bone defect caused by injury, disease, wounds, or surgeryutilizing an implantable mesh of this disclosure comprising acombination of fibers of demineralized bone matrix obtained fromallograft bone, and fibers of non-allograft bone material, the fibers ofnon-allograft bone material comprising non-fibrous demineralized boneparticles embedded within or disposed on the fibers of non-allograftbone material, all mechanically entangled into the implantable mesh.

It should be understood that the forgoing relates to exemplaryembodiments of the disclosure and that modifications may be made withoutdeparting from the spirit and scope of the disclosure as set forth inthe following claims.

What is claimed is:
 1. An implantable mesh comprising a plurality ofbiodegradable polymer mesh fibers mechanically entangled by needlepunching with a plurality of demineralized bone fibers to form a needlepunched implantable mesh, the polymer mesh fibers being a base meshlayer having the demineralized bone fibers disposed within the base meshlayer by needle punching such that the demineralized bone fibers aremechanically entangled into the base mesh layer along a longitudinalaxis of the implantable mesh; the biodegradable polymer mesh fiberscomprising a bioerodible polymer having a molecular weight from about1,000 Daltons to about 30,000 Daltons, the plurality of demineralizedbone fibers comprising from about 20% to about 60% by weight of theimplantable mesh, wherein the implantable mesh does not contain acarrier or an adhesive.
 2. An implantable mesh of claim 1, wherein theneedle punched implantable mesh comprises a plurality of layers in theimplantable mesh.
 3. An implantable mesh of claim 1, wherein (i) themesh is porous having pores from about 100 μm to about 200 μm; or (ii)the mesh is woven, non-woven, knitted, wrapped, plied, braided or amixture thereof.
 4. An implantable mesh of claim 1, wherein the meshcomprises (i) natural materials, synthetic polymeric resorbablematerials, synthetic polymeric non-resorbable materials or mixturesthereof; or (ii) carbon fiber, metal fiber, polyetheretherketones,non-resorbable polyurethanes, polyethers, polyethylene terephthalate,polyethylene, polypropylene, Teflon or mixtures thereof.
 5. Animplantable mesh of claim 4, wherein the synthetic polymer resorbablematerials comprise poly(lactic acid) (PLA), poly(glycolic acid) (PGA),poly(lactic acid-glycolic acid) (PLGA), polydioxanone, PVA,polyurethanes, polycarbonates, polyhydroxyalkanoates(polyhydroxybutyrates and polyhydroxyvalerates and copolymers),polysaccharides, polyhydroxyalkanoates polyglycolide-co-caprolactone,polyethylene oxide, polypropylene oxide, polyglycolide-co-trimethylenecarbonate, poly(lactic-co-glycolic acid) or mixtures thereof.
 6. Animplantable mesh of claim 4, wherein natural materials comprise silk,extracellular matrix, demineralized bone fibers, collagen, ligament,tendon tissue, silk-elastin, elastin, collagen, cellulose or mixturesthereof.
 7. An implantable mesh of claim 1, wherein the demineralizedbone fibers have (i) an aspect ratio of from about 50:1 to about 1000:1;(ii) a diameter from about 100 μm to about 2 mm; or (iii) a length fromabout 0.5 cm to about 10 cm.
 8. An implantable mesh of claim 1, whereinthe implantable mesh further comprises a plurality of surfacedemineralized bone fibers, bone chips, bone particles or mixturesthereof inside the implantable mesh.
 9. An implantable mesh of claim 2,further comprising (i) an osteinductive and/or osteopromotive additiveincluding a bone marrow aspirant, blood, a blood product, a bonemorphogenetic protein, a growth factor disposed on the biodegradablemesh fiber, (ii) a therapeutic agent or mixtures thereof; or (iii)collagen fibers mechanically entangled into the mesh.
 10. An implantablemesh of claim 1, wherein the bioerodible polymer is collagen.
 11. Animplantable mesh of claim 1, further comprising one or more syntheticresorbable ceramics having a tricalcium phosphate: hydroxyapatite weightratio of about 50:50 to about 95:5.